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Biomedical subjects

B Mattiasson

Publications and source records attributed to B Mattiasson.

At least 91 records · Page 5Linked to original sources
PubMed1997

The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can be improved by optimisation of the temperature and by using a high co-substrate concentration.

The stereoselective reduction of ketones by alcohol dehydrogenase from Thermoanaerobium brockii was studied in organic reaction media. 2-Propanol was used as co-substrate to regenerate the coenzyme NADPH. The enantiomeric excess of the alcohol formed from the ketone decreased during the course of the reaction (from 53 to 0% e.e. in the formation of (R)-2-butanol). This was interpreted as being due to the reversibility of all the reactions involved. By using a large excess of 2-propanol this effect was suppressed. In the reduction of 2-butanone to (R)-2-butanol, the enantiomeric excess increased with increasing temperature, but in the reduction of 2-pentanone to (S)-2-pentanol the enantiomeric excess decreased with increasing temperature. The data were evaluated in terms of free energy of activation of the reaction pathways leading to the different possible products.

1-Propanol↗
PubMed1997

Minimal-Fouling enzyme electrode for continuous flow measurement of whole blood lactate.

A new membrane system evaluated in an enzyme electrode for monitoring of whole blood lactate under continuous flow conditions has been developed. The membrane confers functional haemocompatibility and has been coupled to an electrode based on amperometric detection of hydrogen peroxide with lactate oxidase interposed between the haemocompatible membrane acting as diffusion barrier and an inner membrane excluding interfering electroactive species. Here, the enzyme electrode is combined with a flow-through measuring chamber and a double lumen catheter permitting continuous sampling and heparinization of blood. The blood contacting membranes have unique haemocompatibility properties and were produced through modification of poly(vinyl chloride), with the nonionic surfactant Pluronic F-68. On-line monitoring of whole blood samples with different amounts of added lactate was carried out to simulate fluctuations in blood lactate levels during extracorporeal monitoring and results were compared with a standard lactate analyser.

Biocompatible Materials↗
PubMed1996

Polyelectrolyte complexes as vehicles for affinity precipitation of proteins.

Polyelectrolyte complexes (PECs) were formed with polyethylene imine (PEI) and polyacrylic acid sodium salt (PA). The aqueous solubility of such PLCs is dependent on the stoichiometry between the polymers, the charge densities of the polymers and salts present in the solution. Cibacron blue 3GA (CB) was coupled to the PEI and the PECs were used for affinity precipitation of lactate dehydrogenase (LDH) in beef heart extracts. The affinity precipitation was induced by a shift in pH, while the desorption and separation of LDH from the PECs was performed by addition of KCl combined with a shift in pH. LDH was obtained with a yield of 85% and a purification factor of approx. 11-fold. The polymers were recovered and reused once and the results became similar. Prior to the affinity precipitation, interfering nucleic acids were removed by precipitation with PEI.

Acrylic Resins↗
PubMed1996

Calorimetric studies on solid alpha-chymotrypsin preparations in air and in organic solvents.

Differential scanning calorimetry was the method to investigate the thermostability of chymotrypsin. The transition temperature decreased by approx. 30 degrees C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbitol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the enzyme was equilibrated through the apolar solvent, isooctane, stabilization of chymotrypsin was observed at high water activities, compared to equilibration in air. The presence of isooctane resulted in a remarkable stabilization of the chymotrypsin-sorbitol complex. A sorbitol concentration of 5 mmol/g of protein was prerequisite to induce stabilization when equilibrated through isooctane at high water activities. The transition enthalpy increased with increasing amounts of sorbitol. Different hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffer salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water activity 0.97. Variations on the hydration of the preparations both offered the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalyst whose hydration was suppressed due to its environment exhibited low enzymatic activity.

Air↗
PubMed1996

Selection of an adhesive phenotype of Streptococcus salivarius subsp. thermophilus for use in fixed-bed reactors.

Streptococcus salivarius subsp. thermophilus was cultivated in a chemostat in order to obtain an adhesive phenotype of this strain. When the system was operated at low dilution rates (D < 0.2 h-1) for about 4 weeks, the strain formed a visible film on the surface of the culture vessel. The biofilm cells were not washed out even when dilution rates were increased (D = 6.9 h-1), and this resulted in a high biomass productivity (P = 4.1 g1(-1) h-1). On the other hand, when the culture was grown at dilution rates faster than 0.2 h-1, only the free suspended cells were present in the culture broth, and were washed out at velocities of about 1.0 h-1. The biomass productivity was consequently lower (P = 1.33 g1(-1) h-1) than in the previous case. The selected adhesive phenotype was grown on different glass beads and the possibility of lactate fermentation in a continuous and semicontinuous mode was demonstrated.

Bacterial Adhesion↗
PubMed1996

Polymer-shielded dye-ligand chromatography of lactate dehydrogenase from porcine muscle in an expanded bed system.

Dye-affinity chromatography is a widely used technique in protein purification. It has recently been shown that the efficiency of the chromatography process can be significantly improved by pretreatment of the affinity matrix with certain water soluble polymers such as poly(vinyl pyrrolidone). This technique termed as polymer-shielded, dye-affinity chromatography has been successfully used in packed bed mode at a lab scale for the purification of a number of enzymes. The present work deals with the application of polymer-shielded dye-affinity chromatography in an expanded bed system of Streamline-Cibacron Blue 3GA for the isolation of lactate dehydrogenase from a crude porcine muscle extract. The elution conditions were optimised to obtain an efficient process. A higher recovery of the target enzyme (78%) was obtained from the polymer shielded column as compared to the unshielded column (17%), after low ionic strength elution. The purification factor obtained after chromatography on the polymer shielded column was higher (4.1) than that from unshielded column (1.8).

Animals↗
PubMed1995

Fatty acid selectivity of a lipase purified from Vernonia galamensis seed.

Vernonia galamensis is an annual herb whose seed oil contains high levels of an epoxy fatty acid, vernolic (cis-12,13-epoxy cis-9-octadecenoic) acid. The seed also contains lipase activity in the dormant state. A lipase was purified from the seed and its substrate specificity studied in isooctane. The lipase shows pronounced selectivity for the native triacylglycerol, trivernolin. The rate of hydrolysis of triolein, the corresponding non epoxy triacylglycerol, is only 3% of that of trivernolin. In the acidolysis of tricaprylin using a mixture of fatty acids, the Vernonia lipase also showed selectivity for vernolic acid. Michaelis-Menten kinetics of the hydrolysis of triacylglycerols revealed that the observed high selectivity of the Vernonia lipase for trivernolin was mainly due to a higher Vmax for trivernolin. The Vmax value for the hydrolysis of trivernolin was 5 times higher than that for triolein. This novel substrate specificity is an adaptation by the seed lipase to the triacylglycerols of the seed oil that contain up to 80% vernolic acid.

Epoxy Compounds↗
PubMed1994

Interaction of Cibacron blue with polymers: implications for polymer-shielded dye-affinity chromatography of phosphofructokinase from baker's yeast.

Interactions between Cibacron Blue F3GA and water-soluble non-ionic polymers were investigated by monitoring the spectral shift that accompanies the binding phenomena. Polyvinylpyrrolidone (PVP) and poly(vinyl alcohol) were the only polymers among those tested found to interact effectively with the dye. The difference spectra for the PVP-dye complex was typical of "electrostatic interaction spectra" at low ionic strength and typical of "hydrophobic interaction spectra" in the presence of 1.5 M KCl. The binding constant and the number of binding sites per polymer molecule were calculated using the simplest model of independent binding sites. One dye molecule was bound by a PVP segment with a molecular mass of 1000-1300. Regardless of the size of the polymer molecules, the binding constants were in the micromolar range. Poly(vinyl alcohol) bound less efficiently to Cibacron Blue than PVP. One dye molecule was bound by a polymer segment with a molecular mass of about 10,000. The data on PVP complexing with Cibacron Blue were used to develop the concept of polymer-shielded dye-affinity chromatography. This concept was successfully applied to the chromatography of phosphofructokinase (EC 2.7.1.11) from baker's yeast. Specific elution of the bound enzyme from PVP-shielded column resulted in an efficient process with 27-fold purification.

Chromatography, Affinity↗
PubMed1994

Integration of aqueous two-phase extraction and affinity precipitation for the purification of lactate dehydrogenase.

Integration of extraction in aqueous two-phase system and affinity precipitation was investigated as a technique for purification of lactate dehydrogenase (LDH) from porcine muscle extract. An enteric coating polymer, Eudragit S 100, which can be made reversibly soluble and insoluble by change in pH was used as the ligand carrier. The ligand used was Cibacron blue 3GA. The polymer is nearly totally partitioned to the top phase (> 98%) in PEG-dextran aqueous two-phase system. The enzyme, lactate dehydrogenase, was first spontaneously partitioned to the bottom phase in a 6% (w/w) PEG 8000-8% (w/w) dextran T250 phase system. New PEG phase and Eudragit-dye were then added to the bottom phase, which helped in extraction of LDH to the top phase. After a washing step with a fresh bottom phase, Eudragit-dye-target protein affinity complex was precipitated out from the top phase by lowering the pH to 5.1. The enzyme was recovered by treatment of the complex with 0.5 M NaCl with a yield of 54% and a specific activity of 245 units/mg. The purification of LDH by this procedure was better than that obtained by a single step of affinity partitioning.

Acrylic Resins↗
PubMed1994

Synthetic polymers as solubilizing vehicles for enzymes in water-poor media.

A recent method for exposing enzymes to organic solvents is reviewed. By complex formation between the enzyme and polymers that per se are soluble in organic solvents it is possible to disperse the enzyme in the organic medium in such a way that an optically transparent (in the visible region) solution is obtained. After reaction, the separation of the enzyme from the organic medium can be obtained simply by addition of water. The enzyme can be recovered from the water phase. Physicochemical studies have revealed that the enzyme is more stable in the complex-bound form.

Cellulose↗
PubMed1993

A microbial biosensor system for dihalomethanes.

A biosensor system able to measure dichloromethane (DCM) and other dihalomethanes has been developed. The analysis is based on Hyphomicrobium DM2 cells immobilized in alginate. A combination of transducers consisting of a flow-calorimeter followed by a chloride-sensitive electrode has been used. By this design it was possible to monitor different aspects of the cell metabolism from one and the same pulse of substrate. The detection limit for the biosensor was 0.1 microM dichloromethane. The biosensor system can be used for continuous measurements in a sample stream.

Bacteria↗
PubMed1993

Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology.

Thermoprecipitating polymers such as poly (N-isopropylacrylamide), poly(N-vinyl caprolactam), and some ethylene oxide-containing surfactants appear to be suitable for developing new separation systems to complement traditional precipitation, chromatography, and extraction of biological molecules. The nature of thermally induced phase separation of polymers and nonionic surfactants is discussed and examples are given. Covalent coupling of an enzyme to a thermoprecipitable polymer results in a biocatalyst which combines the qualities of soluble and immobilized enzymes. Biocatalysts of this type can be separated from reaction media by precipitation after temperature increase. The use of thermoprecipitating polymer-protein conjugates in immunoassays overcomes one of the shortcomings of traditional methods with solid sorbent-linked antigen or antibody-diffusional limitations. Thermoreactive hydrogels produced by crosslinking of thermoprecipitating polymers can be successfully used for concentrating macromolecules or microbe-rich slurries. Alternate volume changes of hydrogels on heating and cooling produce a "hydraulic pump" which can enhance the productivity of an immobilized biocatalyst. Hydrogels could be used to control reaction or diffusion rates by a thermal feedback mechanism.

Biotechnology↗
PubMed1993

Sampling and sample handling--crucial steps in process monitoring and control.

Sampling and sample handling are important considerations in the design of monitoring and control systems for bioreactors. Many factors must be considered in developing strategies for obtaining representative samples, thus ensuring that the status of the bioprocess can be evaluated rapidly, in a reproducible and reliable manner.

Biotechnology↗
PubMed1993

Automated immunochemical binding assay (flow-ELISA) based on repeated use of an antibody column placed in a flow-injection system.

A computer-controlled immunological binding assay intended for monitoring concentrations of interesting metabolites during bioprocesses has been developed. The main focus has been on speed, reliability, reproducibility and operational stability of the assay. The assay is based on the repeated use of a preparation of immobilized antibodies. Stability in the regeneration step is a prerequisite and was achieved by intermittent recalibration of the system. Furthermore, before immobilizing the polyclonal antibodies, they were purified on immobilized antigen and eluted using the same procedure as used in the assay cycle. Antibody preparation could be used for more than 50 cycles and in some cases for several hundred cycles. Since the calibration curve is valid for the whole life-time of the antibody column, recalibration only involved the registration of the column capacity. Experiments have been performed concerning monitoring of chromatographic separations as well as adsorption experiments from complex mixtures.

Animals↗
PubMed1993

Portable system for continuous ex vivo measurements of lactate.

The lactate profile in serum was monitored continuously in volunteers under physical exercise on a test bicycle. The assay was operated ex vivo by means of continuous sampling through a coaxial catheter. After dialysis online, lactate was converted by means of lactate oxidase immobilized to porous glass, and the depletion of oxygen was registered by means of a Clark electrode. Good agreement between the continuous monitoring and off-line analyses was found.

Biosensing Techniques↗